Expression of heterologous proteins in yeast after transformation of yeast cells with suitable expression vectors comprising DNA sequences coding for said proteins has been successful for many species of polypeptides, such as insulin precursors, glucagon, glucagon like peptides and their functional analogues.
However, it is often found that the expression product is a heterogeneous mixture of species of the desired polypeptide precursor having different amino acid chain lengths. This is because yeast produces a number of proteolytic enzymes being responsible for processing of larger precursor molecules to release the mature polypeptide. A number of proteases including the PEP4 and KEX1 gene products are responsible for such yeast protein degradation.
The use of KEX1 disrupted strains for expression of recombinant proteins have been described earlier. Thus EP341215 and U.S. Pat. No. 6,103,515 describe use of a yeast strain which lacks KEX1 for production of peptides which bear no basic C-terminal amino acid e.g. hANP, EGF, connective tissue activating peptide-III hirudin and hirudin variants.
More than half of the known neural and endocrine peptides are α-amidated and in most cases, this structural feature is essential for receptor recognition, signal transduction, and thus, biologic function. The α-amidation is derived from a C-terminal glycine that is enzymatically converted to an amide.
An α-amidated peptide may be produced directly in the production cell if the cell has the necessary machinery for in vivo α-amidation. However, some organisms including yeast are not capable of making the α-amidation because they do not express the necessary enzyme for in vivo α-amidation and therefore peptides produced by the cell have to be α-amidated by a subsequent in vitro step.
If yeast is used as the recombinant production cell one solution is to produce a precursor polypeptide with a C-terminal glycine which is then in a subsequent in vitro process converted to the α-amidated peptide using an α-amidating enzyme (D. J. Merkler (1994). C-Terminal amidated peptides: Production by the in vitro enzymatic amidation of glycine extended peptides and the importance of the amide to bioactivity. Enzyme Microb. Technol.: 16(450-456).
Surprisingly however, the inventors of the present invention have discovered that the C-terminal glycine is cleaved off from a number of peptides expressed from Saccharomyces cerevisiae making the subsequent conversion to an α-amidated peptide impossible.
The present invention offers a solution to this problem by using a genetically modified yeast strain which will not cleave off the C-terminal glycine residue.